Current models of cardiac electrophysiology and arrhythmia focus on the electrical properties of the cardiomyocyte. Recent investigations, though, have begun to establish remarkable roles for non-myocyte cell types, including ?broblasts and immune cells, in cardiac conduction and electrical remodeling after injury. The concept that conduction in the heart depends upon multiple cell types has the potential to alter many of our current hypotheses about arrhythmia generation and promises entirely new approaches to therapy. Revolutionary advances in microscopy, transgenic mouse models, ?uorescent reporters, and optogenetics tools are fueling paradigm shifts in our understanding of complex cellular neuronal physiology in the brain. Yet investigations into cellular connectivity in the heart to date have depended upon in vitro examinations of electrophysiology (patch clamping, co-culture) or low resolution readouts of conduction (ECG, conventional optical mapping). This Trailblazer R21 application proposes to overcome limitations of standard optical mapping approaches by creating a new platform for in vivo all-optical electrophysiology studies in the beating heart at cellular resolution. Aim 1 proposes to combine high resolution cardiac intravital microscopy techniques with multiplexed ?uorescent reporters and cell-speci?c optogenetics actuators to enable simultaneous optical mapping and stimulation of electrical activity in the mouse heart with cellular resolution. Aim 2 will then apply this platform to study the electrical connectivity of myocytes and non-myocytes during healing after myocardial infarction. If successful, this application will signi?cantly advance the ?elds of cardiac imaging and electrophysiology and will address key questions with clinical implications about electrical remodeling and models of arrhythmia generation after myocardial infarction.